This invention relates to a large DNA cloning system and, more particularly, to vectors that allow the cloning of large segments of exogenous deoxyribonucleic acid (DNA) as yeast artificial chromosomes.
The baker's yeast Saccharomyces cerevisiae is one of the most useful eukaryotic organisms in the field of molecular biology. It has a small genome or DNA content only about 3 times that of E. coli, a short generation time of a few hours and it is as readily manipulated as most procaryotes, yet it exhibits some very complex phenomena specific to eukaryotes such as chromosome structure, mitotic and meiotic cell division.
With the advent of recombinant DNA and molecular cloning technology, it is now possible to transfer genetic information from any source into microorganisms, including yeasts. Using conventional recombinant DNA techniques, small plasmid and viral chromosomes can be constructed in vitro and then transferred into host cells and clonally propagated.
Most DNA cloning systems have a capacity only for relatively small segments of exogenous DNA, for example, on the order of up to about 50 kilobases (kb), and bacterial clones such as E. coli are frequently unstable. These cloning systems are well suited to the analysis and manipulation of typical genes and small gene clusters, particularly from organisms in which the genetic information is tightly packed. It is increasingly apparent, however, that many of the functional genetic units in higher organisms span enormous tracts of DNA. For example, the bithorax locus in Drosphila, which is involved in regulating the development of the fly's segmentation pattern, encompasses approximately 320 kb [Karch et al., Cell 43, 81 (1985)]. The factor VIII gene in the human, which encodes the blood-clotting factor deficient in hemophilia A, spans 190 kb [Gitschier et al., Nature 312, 326 (1984)]. Recent estimates of the size of the gene that is defective in Duchenne's muscular dystrophy suggest that this single genetic locus, whose protein-coding function could be fulfilled by as little as 15 kb of DNA, actually covers more than a million base pairs [Monaco et al., Nature 323, 646 (1986)].
It is thus seen that a DNA cloning system that allows the cloning of large segments of exogenous DNA, on the order of greater than 50 kb, would have significant utility.
Although techniques exist for cloning large genes or gene clusters in many overlapping, relatively small pieces, this process is laborious, error-prone, and poorly suited to functional studies of the cloned DNA. Furthermore, there are a number of problems in molecular genetics that will require the characterization of even more extensive tracts of DNA than those present in the largest known genes. For example, the regulated somatic DNA rearrangements that give rise to functional immunoglobulin and T-cell-receptor genes involve deletions of whole segments of chromosomes, while some of the genetic events that have been implicated in the induction or progression of malignant tumors involve the amplification of similarly large regions. See Tonegawa, Nature 302, 575 (1985); Kronenberg et al., Ann. Rev. Immuno. 4, 529 (1986); and Bishop, Science 235, 305 (1987). In other instances, including efforts to define the primary defects in such genetic diseases as Huntington's chorea and cystic fibrosis, only the genetic linkage between the closest identified clones and the disease locus is known. See Beaudet et al., Am. J. Hum. Genet. 39, 681 (1986), and Gusella et al., Nature 306, 234 (1983). In typical cases, the search for the locus itself will require the analysis of megabase-pair regions of DNA. Mapping of these large regions of DNA can lead to the cloning of candidate genes. Isolation of the aberrant gene will facilitate development of better markers and probes for the gene and this, in turn, can result in diagnostic tests for the genetic disease. Ultimately, isolation and characterization of the defective gene can lead to the development of therapeutic interventions.
Finally, there is increasing interest in the global mapping of the DNA of intensively studied organisms. See Olson et al., Proc. Natl. Acad. Sci. USA 83, 7826 (1986), and Coulson et al., Ibid., 83, 7821 (1986). See also the reports of discussions by the National Academy of Sciences to map and sequence the human genome in Science 235, 747-748 (1987). Particularly in the case of the human, or other organisms with comparably complex genomes, such projects would require the ordering of hundreds of thousands of conventional clones. A cloning system that allowed the same objective to be achieved with many fewer clones would not only improve mapping efficiency, but it would also have dramatic effects on the reliability and continuity of the final map.